Abstract In this application, we propose several studies to substantially improve our understanding of the role of oxidative stress and antioxidants in breast carcinogenesis. Circulating levels of fluorescent oxidation products, an innovative marker of overall oxidative stress, have been associated with an increased risk of cardiovascular disease, but to our knowledge, have not been investigated in relation to breast cancer risk. Carotenoids, prominent micronutrients in fruits and vegetables, have been of specific interest in breast cancer because of their antioxidant properties. Although findings from epidemiologic studies of dietary intake have been mixed, recent data have shown a protective effect of plasma carotenoid levels on breast cancer risk. Specifically, 5 of 6 prospective studies of plasma carotenoids, including our own previous work in the Nurses'Health Study (NHS), found significant or suggestive inverse associations with breast cancer risk. However, the specific carotenoid(s) associated with risk has not been consistent across studies, the importance of timing has not been well addressed, and subgroup analyses have been underpowered. Finally, we propose to examine the association between breast cancer risk and variation in antioxidant enzyme and carotenoid metabolism genes, and the interactions between these genes, as well as DNA repair genes, and the plasma markers. To our knowledge only a prior initial analysis of 1 of these genes within the NHS has investigated the interactions with plasma carotenoids, and none has examined the interactions with oxidative stress. Within the NHS cohort, with archived blood samples and a wealth of data on breast cancer risk factors, we propose an in depth, prospective investigation of plasma markers of oxidative stress, plasma carotenoids, and genetic variation. In this study, we will utilize archived (stored at d -1300C) blood samples collected in 1989-1990 from 32,826 women in the NHS;18,743 of these women donated a 2nd sample in 2000. Our nested case-control design will efficiently utilize these prospectively collected samples. A unique strength of this study is the availability of 2 measures collected 10 years apart to examine the timing of exposure in the pathogenic process among approximately 700 cases confirmed after the 2nd blood collection through 2008. In our carotenoids analyses, utilizing our prior data, we will have approximately 2000 cases diagnosed 1990-2008 to more thoroughly assess specific carotenoids and conduct important subgroup analyses. Similarly, in the genetic analyses we will have over 2000 cases to address the main effects of genetic variation in antioxidant and carotenoid metabolism genes, as well as interactions between these genes, DNA repair genes, and the plasma markers. This study provides an ideal context in which to investigate these associations, given the prospective nature, extensive covariate information, and an ongoing, high rate of follow-up (98% in the blood cohort). Our proposed study should contribute substantially to our understanding of the relationships between the oxidative stress pathway and breast cancer risk and the role of antioxidants in protecting against oxidative damage. PUBLIC HEALTH RELEVANCE: Oxidative stress may play an important role in the development of breast cancer through its ability to damage protein and DNA while antioxidants, from both enzymes within the body and external dietary sources, may counteract this potentially harmful pathway. This study will examine the relations of plasma markers of oxidative stress, plasma levels of carotenoids, and genetic variation in related genes with the risk of breast cancer in the prospective Nurses'Health Study cohort. Confirmation of a detrimental effect of oxidative stress and the protective benefit of carotenoids, which are amenable to dietary change, would have important public health implications.